Disease modeling

iPSC technology has enabled the creation of in vitro disease models in which disease-specific hiPSCs from patient somatic cells or differentiated into damaged cell types by editing disease-causing genes in hiPSCs from healthy donors to mimic the disease phenotype (Figure 1a). earlier work by Freedman et al. from autosomal dominant PKD1 gene mutations caused by polycystic kidney disease (ADPKD) patients generated hiPSCs and found that hiPSCs and their differentiated cells exhibited downregulation of polycystin 2, elucidating a novel mechanism by which the PKD1 gene encoding polycystin 1 regulates polycystin 2 expressions. Our group cultured hiPSCs from patients with ADPKD, including those with combined intracranial aneurysms, and confirmed that intracellular calcium handling and expression of extracellular matrix-related genes were altered in vascular cells differentiated from hiPSCs, consistent with vascular cells from a mouse model of ADPKD and renal cyst cells from patients with ADPKD.

Figure 1：Modeling ADPKD using disease-specific hiPSCs. a: A schematic showing disease modeling research for ADPKD. Disease-specific hiPSCs are derived by reprogramming the somatic cells of ADPKD patients or gene editing PKD1/2 in hiPSCs derived from healthy donors. Disease models are generated by differentiating the theADPKD-specific hiPSCs into renal tissues for pathologic analysis and drug discovery

The latest advances in generating renal unit-like organs from hiPSCs have enabled the modeling of renal diseases such as renal nephropathy, congenital nephrotic syndrome, and autosomal recessive polycystic kidney disease (ARPKD). Models of ADPKD renal cysts using renal unit-like organs have been generated from gene-edited pure-sibling pkd1 /2 mutant hESCs. However, when using ADPKD patient-derived or gene-edited heterozygous pkd1 mutant hiPSCs, these models did not generalize the renal cyst phenotype seen in ADPKD. In contrast, we recently generated renal unit-like organs from ADPKD patient-derived and gene-edited heterozygous and pure pkd1 mutant hiPSCs by forskolin treatment to propagate renal cysts. Notably, we confirmed that all three hiPSC types can form renal cysts (Figure 1b-d). These renal cysts responded to some drugs known to inhibit cyst formation in ADPKD, such as the mammalian target of rapamycin (mTOR), suggesting that these models could be used to screen for drug compounds to prevent renal cyst formation (Figure 1e). We are currently developing a high-throughput chemical screening system for therapeutic drug discovery in ADPKD by modifying the cyst model.

Figure 1: b: Representative bright-field images of wild-type and gene-edited PKD1-mutant hiPSC-derived kidney organoids after 7 days of forskolin treatment. c: Quantification of the cystic areas of the kidney organoids in (b). Data are represented as the mean±SE from three independent experiments with four replicates in each. **p<0.005 and ***p<0.001 by one-way ANOVA and Bonferroni’smethod. d: Representative bright-field images of the normal subject- and ADPKD patient-derived kidney organoids after 7 days of forskolin treatment. e: Representative bright-field images of patient-derived kidney organoids after 7 days of treatment with CFTR inhibitor 172 (100 μM) or everolimus (10 μM) in the presence of forskolin. Scale bars, 300 μm in (b), (d, right), and (e) and 500 μm in (d, left). Adapted from Shimizu et al.

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Addressing the complications of kidney diseases

Major complications associated with chronic kidney disease (CKD) include renal anemia caused by inadequate production of the renal hematopoietic hormone erythropoietin (EPO). Although renal anemia has been successfully treated by intermittent administration of recombinant human EPO preparations, additional physiological therapies are needed. Considering that EPO is also produced in the liver during embryonic or adult severe anemia, we modified the previously reported liver differentiation protocol to successfully produce EPO cells from hiPSCs (hiPSC-EPO cells). These hiPSC-EPO cells upregulate EPO production in response to hypoxic stimulation, similar to their in vivo counterparts. Based on colony formation assays using human hematopoietic progenitor cells, EPO proteins contained in culture supernatants showed differentiation-promoting effects on the red lineage. Furthermore, these hiPSC-EPO cells improved renal anemia for 7 months after transplantation in an adenine-induced mouse model. Thus, hiPSC-EPO cells could be used to discover new drugs and develop cellular therapies for the treatment of renal anemia.

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Cell therapy

The use of hiPSC-derived embryonic kidney progenitor cells for cell therapy has been investigated. To investigate their therapeutic potential for renal diseases, we transplanted NPC-like renal progenitor cells generated from hiPSCs into the subcapsular of a mouse model of acute kidney injury (AKI) induced by ischemia/reperfusion injury using our differentiation method and found that transplantation significantly suppressed the elevation of blood urea nitrogen (BUN) and serum creatinine (Cre) in the host mice. In addition, treatment significantly ameliorated AKI-induced histological damage, such as tubular necrosis. Notably, interstitial fibrosis, which reflects chronic disease progression, was also significantly prevented. Transplanted progenitor cells improved AKI without integration into host kidney tissue, suggesting that the paracrine effects of renal trophic factors secreted by hiPSC-derived renal progenitor cells are largely responsible for the therapeutic benefit. Elucidation of these factors will help in the development of anti-AKI cellular therapies and novel drugs.

Imberti et al. also reported the therapeutic benefit of using hiPSC-derived renal progenitor cells to treat a mouse model of cisplatin-induced AKI. They injected hiPSC-derived NPC-like renal progenitor cells into the AKI mouse model through the tail vein. This transplantation treatment also significantly improved AKI, as evidenced by reduced BUN levels and histological findings.

Although the differentiation protocol for generating renal progenitor cells differs from that of the AKI mouse model, these two reports demonstrate for the first time the potential therapeutic benefit of using hiPSC-derived renal progenitor cells for the treatment of kidney disease.

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Conclusion and future perspective

Substantial progress has been made in generating embryonic kidney progenitor cells and kidney tissue from hPSCs. However, there are still some obstacles to overcome before clinical application. Regarding renal reconstruction, the generation of larger renal tissues and pelvis and ureter-like structures, in which collecting ducts aggregate, has not been achieved. In addition, the integration of hiPSC-derived renal structures with large blood vessels is required. Cell therapy using hiPSC-derived renal progenitor cells should also be examined in a CKD model. Finally, hiPSC-based models have been developed for a number of renal diseases including ADPKD, and are expected to identify drug-candidate compounds against renal diseases.

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